Mechanical Response of Neural Cells to Physiologically Relevant Stiffness Gradients

Abstract

Understanding the influence of the mechanical environment on neurite behavior is crucial in the development of peripheral nerve repair solutions, and could help tissue engineers to direct and guide regeneration. In this study, a new protocol to fabricate physiologically relevant hydrogel substrates with controlled mechanical cues is proposed. These hydrogels allow the analysis of the relative effects of both the absolute stiffness value and the local stiffness gradient on neural cell behavior, particularly for low stiffness values (1-2 kPa). NG108-15 neural cell behavior is studied using well-characterized collagen gradient substrates with stiffness values ranging from 1 to 10 kPa and gradient slopes of either 0.84 or 7.9 kPa mm^-1 . It is found that cell orientation is influenced by specific combinations of stiffness value and stiffness gradient. The results highlight the importance of considering the type of hydrogel as well as both the absolute value of the stiffness and the steepness of its gradient, thus introducing a new framework for the development of tissue engineered scaffolds and the study of substrate stiffness.

Keywords: mechanosensitivity; neurites; peripheral nervous system; stiffness gradients.

Figure 1

a) Schematic diagram showing the fabrication process of the collagen gradient gel mould with defined geometry were CAD‐designed and 3D printed using PLA. Collagen gel solution was pipetted in moulds and gels were left to set for 15 min in a humidified incubator and then flipped to a standard 24‐well plate. Then, gels were RAFT‐stabilized for 15 min. The gels are segmented into three separate regions (I–II–III) from soft to stiff. b) Photographs of the experimental setup to make gradients gels.

Figure 2

a) Spatial mapping of the elastic modulus of Control gel and Lower and Higher gradient gels. The elastic modulus at each pixel is the mean of five measurements taken by AFM indentation, and is represented as a color map with blue denoting softer (0 kPa) and yellow corresponding to stiffer (8 kPa) regions. Stiffness distributions (E, kPa), for each of the three segment (I–II–III) of the Lower and Higher gels were statistically analyzed. N = 56 per segmented area for the Lower gradient and N = 49 per segmented area for the Higher gradient (one‐way ANOVA test and Tukey–Kramer multiple comparison's test). Asterisks indicate significant statistical differences (*P < 0.05, ****P < 0.0001). b) Mean Young's modulus averaged for 3 gels every 125 µm over a 2.5 mm distance (corresponding to 20 segments) and corresponding gradient slope (Control (0%), Lower (2.5%), and Higher (45%)).

Figure 3

AFM topography images (10 × 10 µm, 1024 × 1024 pixels) of RAFT‐stabilized collagen gels for the Control gel, and the three segments (I–II–III) of the Lower and Higher gels. The first two columns show the 3D and 2D view of the relative value of the height of the gels (scale bar in nm). The third column is the error signal (mV) indicating the surface of the collagen gels where the banding pattern of collagen type I fibrils is visible. The surface roughness of the gels is indicated by the height profiles in the last column.

Figure 4

Representative fluorescence micrographs showing NG108 cells after 48 h in culture on top of gradient or Control gels. Nuclei were labeled with Hoescht (blue) and β‐III‐tubulin immunoreactivity (green) was used to detect neurites on each part (I–II–II) of the Lower and Higher gradient gels, indicated by the white arrows. White scale bars: 100 µm.

Figure 5

Quantification of neurite response to gradient gels. NG108‐15 cells were cultured on two different gradient slopes (Lower and Higher) and on Control gel for 48 h. a) Fluorescence micrographs show sprouting of neurites; red dashed line indicates neurite length, branching spots are indicated by red arrows, and neurite orientation was measured as indicated by the white line. The white arrow indicates the direction of the gradient. Schematic indicates the classification of neurite orientation: neurites were considered as growing up the gradient for angles between −60° and 60° (yellow), down the gradient for the angles between −120° and 120° (blue), and perpendicular to the gradient otherwise (gray). Measurements were performed for each gradient segment (I–II–III) of three separate gels, for each condition (N = 3, n = 3). b) Percentage of elongation toward a given direction for each segment of the Control and gradient gels. c) Mean number of neurites per cell, d) mean neurite growth rate (µm h⁻¹), e) percentage of NG108‐15 cells presenting neurites, and f) mean neurite length (µm) do not vary according to the presence of a gradient or differences in absolute stiffness value. However, g) the extent of neurite branching (%) is affected by the stiffness gradient. Data are shown for the different segments (I–II–III) of each gradient type (Lower and Higher) and the Control. Results are shown as mean ± SD (one‐way ANOVA test and Tukey–Kramer Multiple's comparison's test, *P < 0.05).

Figure 6

Graphical summary of the gradients and materials used to study durotactic behavior in vitro, showing the Young's Modulus (kPa) as a function of the location on the samples (mm). Studies are categorized by material used, with the majority using coated PAAm gels as it offers the possibility to work with a wide range of stiffnesses using a large variety of techniques., , , , , This summary includes only 2D cultures. The study of durotaxis is challenging, as it is difficult to uncouple the stiffness of a substrate from its pore size, ligand molecule coating density, and height of the substrate itself. Here, we use collagen type I gels to develop defined stiffness gradients, represented by the red lines (Lower and Higher), with consistent stiffness properties to those explored in the literature.